For measurement, evaluation, and control of a biological reaction such that cells adhere to a biological substrate material in a culture fluid and then grow and differentiate, a phenomenon of hydration of biomolecules, biotissues, biological substrate materials, etc. is important. A hydration structure hereof exhibits a three-dimensional structure which is formed by interaction between the surface of a specimen and water molecules and interaction between water molecules including hydrogen bonding at a specimen-culture fluid interface in a culture fluid composed mainly of water. So-called biocompatibility such as typified by adhesion of red blood cells to the inner wall of an artificial blood vessel is thought to be closely related to this hydration structure (e.g., Non-Patent Literature 1). Moreover, inter alia, irregularities of the surface of a specimen in a culture fluid, potential distribution, and composition distribution and array structure of molecules, proteins, etc. are important characteristics especially with regard to biological reactions of biomolecules, biotissues, biological substrate materials, etc. in a culture fluid.
For specimens of biomolecules, biotissues, biological substrate materials, etc. in a culture fluid, as means for observing the specimen-culture fluid interface and taking measurements, optical microscopes and non-linear optical microscopes based on Raman spectroscopy, second harmonic generation spectroscopy, sum-frequency generation spectroscopy, etc. have so far been used. In particular, the sum-frequency generation spectroscopy enables measurement of array structure of water molecules with regard to the hydration structure at the specimen-culture fluid interface. As a non-liner optical microscope, for example, Patent Literature 1 discloses a surface selective and non-linear optical method for observing interaction between a probe and a target by means of second harmonic light and sum-frequency light emitted by water molecules, solvent molecules, or markers in the vicinity of the interface. However, the spatial resolution of these optical microscopes and non-linear optical microscopes is larger than 100 nm and, typically, on the order of 1 μm.
Meanwhile, scanning probe microscopes are based on Atomic Force Microscopy (AFM). A scanning Kelvin probe microscope, which is an example of a scanning probe microscope, uses the following method: while the microscope detects static electric field force exerted between a cantilever having an electrically-conductive probing needle and a specimen in terms of flexure of the cantilever, the probing needle scans the surface of the specimen, thereby mapping distribution of the static electric field force. In addition to static electric field force, atomic force or the like is also applied to the probing needle and it is necessary to separate static electric field power from other interactions. For this reason, the cantilever is first oscillated and the distance between the probing needle and the specimen is adjusted so as to keep constant the oscillation amplitude that is decreased by atomic force exerted when the probing needle contacts with the specimen. Thereby, the position of the specimen surface in a height direction is determined. The probing needle is set apart from that position, i.e., the specimen surface at a certain distance and, in this condition; static electric field force which is long-range force is detected from phase change of the oscillation of the cantilever (e.g., Patent Literature 2). In terminology of scanning probe microscopes, the probing needle may be called a probe.
With scanning probe microscopes, spatial resolution of on the order of 1 nm for irregularity measurement and spatial resolution of on the order of 10 nm for static electric field and optical measurement can be expected. However, because a region of interaction between the probing needle and the specimen is restricted to approximately the diameter of the tip of the probing needle, it is generally difficult to realize a scanning probe microscope using a physical quantity of a weak signal, particularly as in the non-linear optical method.
Literature concerning a tip-enhanced Raman detection method that uses surface-enhanced Raman scattering by a probing needle to compensate this weak signal and realize Raman spectroscopy with a scanning probe microscope is disclosed. Patent Literature 3 discloses a probe for a near-field microscope in which the probe is coated reproducibly with uniform metal particles to efficiently induce surface-enhanced Raman scattering in a near-field microscope that inserts the probe into an evanescent field which is generated on the surface of a sample, scatters the evanescent field by the probe tip, and detects scattering light.
Non-Patent Literature 2 sets forth that irradiating a metallic probing needle whose tip has a nanometer diameter with light makes it possible to trap the light in the nano space of the tip and further irradiating it with molecules as a nano light source enables detection of Raman scattering light from the molecules at a nano spatial resolution. This literature also sets forth that, especially, localized plasmon polariton which is a resonance phenomenon is excited at the tip of the probing needle and enhances electric field strength of light, which effectively increases the scattering cross-section of Raman scattering and compensates weak scatterability (tip-enhanced Raman detection method), and a spatial resolution of 15 nm is realized.
Patent Literature 4 discloses an ultraviolet near-filed optical microscope using a tip-enhanced Raman detection method in which the microscope focuses laser light on a specimen to generate Raman scattering light, brings a probing needle close to or in contact with the specimen so that the tip will enhance and scatter the Raman scattering light, and detects a Raman spectrum from the scattering light which has been scattered. In this regard, excitation laser light is ultraviolet or deep ultraviolet laser light and the material of the tip of the probing needle is a metal having a permittivity of −2 or less at an excitation laser wavelength; it is mentioned that, for example, preferable is an aluminum thin film which was vacuum deposited on the surface of the probing needle made of silicon, in which the film thickness is about 25 nm and metal particles are 10 to 20 nm in diameter.
Patent Literature 5 discloses that, using a near-field optical microscope that, while scanning a specimen by a probe (probing needle), detects near-field light that is generated in the vicinity of the probe by light irradiation, it is possible to observe a change in minute oscillation of molecules adsorbed onto the surface, depending on distance between the probing needle and the specimen, as a change in Raman scattering. In this regard, the light source is, inter alia, a laser such as He—Cd (with a wavelength of 441 nm) and, as the probing needle, a 35 nm silver evaporated probe is used.
Patent Literature 6 discloses a technique for holding a maximum value of a light signal that is detected in sync with a laser in an optical probe microscope.
However, in these literatures, neither a scanning probe microscope using second harmonic generation spectroscopy or sum-frequency generation spectroscopy, nor a scanning probe microscope with high spatial resolution by means of Raman spectroscopy is disclosed.